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- PDB-8hsg: Thermus thermophilus RNA polymerase elongation complex -

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Basic information

Entry
Database: PDB / ID: 8hsg
TitleThermus thermophilus RNA polymerase elongation complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • DNA (27-MER)
  • DNA (37-MER)
  • RNA (5'-R(P*CP*CP*AP*CP*AP*UP*CP*CP*AP*CP*CP*U)-3')
KeywordsTRANSCRIPTION / Transcription elongation complex / RNA polymerase
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit ...: / DNA-directed RNA polymerase subunit beta', hybrid domain / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
DNA molecule (others)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMurayama, Y. / Ehara, H. / Sekine, S.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP17K15082 Japan
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from .
Authors: Yuko Murayama / Haruhiko Ehara / Mari Aoki / Mie Goto / Takeshi Yokoyama / Shun-Ichi Sekine /
Abstract: Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the ...Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The β-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.
History
DepositionDec 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 3, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
T: DNA (37-MER)
R: RNA (5'-R(P*CP*CP*AP*CP*AP*UP*CP*CP*AP*CP*CP*U)-3')
N: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)530,54010
Polymers530,4508
Non-polymers902
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHIJK

#1: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 35056.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: rpoA, TTHA1664 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SHR6, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125436.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: rpoB, TTHA1813 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8RQE9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 171994.391 Da / Num. of mol.: 1 / Mutation: C-terminal FLAG-tagged
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: rpoC, TTHA1812 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8RQE8, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11491.237 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: rpoZ, TTHA1561 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8RQE7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules TN

#5: DNA chain DNA (37-MER)


Mass: 57785.004 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#7: DNA chain DNA (27-MER)


Mass: 54576.797 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules R

#6: RNA chain RNA (5'-R(P*CP*CP*AP*CP*AP*UP*CP*CP*AP*CP*CP*U)-3')


Mass: 39054.004 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 2 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RNA polymerase elongation complex / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219822 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 125.42 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002625227
ELECTRON MICROSCOPYf_angle_d0.593134450
ELECTRON MICROSCOPYf_chiral_restr0.0423906
ELECTRON MICROSCOPYf_plane_restr0.00474246
ELECTRON MICROSCOPYf_dihedral_angle_d15.34639836

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